GENOMIC (gDNA) and PLASMID (pDNA)
The nature and purpose of analysis to be carried out affects the DNA of interest. So it is important to understand their difference and extraction methods.
Genomic DNA provides all the information for regular well-being of the cell, while plasmid DNA provides extra characteristics to a cell, like antibiotic resistance, which can allow a cell to survive in harsh conditions. A cell can survive without plasmid DNA, but never without genomic DNA.
Plasmid DNA is naked double stranded DNA that forms a circle with no free ends. It is associated with few protein but contains RNA polymerase enzyme. … Chromosomal (genomic) DNA is double stranded linear DNA molecular associated with large proteins.
Genomic DNA is DNA that is part of an organism’s chromosomes. A Plasmid is small loop of DNA which is separate. Bacteria typically have one circular chromosome, but a variable number of plasmid which they swap with other bacterial of their type. To further itemize it their difference, thus;
1. Genomic DNA
- The genomic DNA of any organism, consists the biological information of heredity which is passed from one generation to the next generation.
- The genomic contains ALL the genes that are needed for the organism for basic survival in normal conditions.
2. Plasmid DNA
- The plasmid DNA is separated from the genomic DNA and known as extrachromosomal DNA.
- It is always covalently closed and circular in form.
- Plasmids contain only additional genes that may be useful to the organism under certain situations or particular conditions.
- They can code for Antibiotic resistant genes, can act as toxins.
- They are used as cloning vectors and expression vectors.
DNA EXTRACTION
Scientists can buy ready-to-use DNA extraction kits. These kits help extract DNA from particular cell types or sample types. However, they can be expensive to use routinely, so many labs have their own methods for DNA extraction.
The ability to extract DNA is of primary importance to studying the genetic causes of disease and for the development of diagnostics and drugs. It is also essential for carrying out forensic science, sequencing genomes, detecting bacteria and viruses in the environment, for determining paternity, molecular analyses including PCR, electrophoresis, sequencing, fingerprinting and cloning etc.
The Basics of DNA Extraction
In order to study DNA, you first have to get it out of the cell. In eukaryotic cells, such as human and plant cells, DNA is organized as chromosomes in an organelle called the nucleus. Most bacterial cells have no nucleus. Their DNA is organized in rings or circular plasmids, which are in the cytoplasm. The DNA extraction process frees DNA from the cell and then separates it from cellular fluid and proteins so you are left with pure DNA.
The three basic steps of DNA extraction are 1) lysis, 2) precipitation, and 3) purification.
Step 1: Lysis
In this step, the cell and the nucleus are broken open to release the DNA inside and there are two ways to do this. First, mechanical disruption breaks open the cells. This can be done with a tissue homogenizer (like a small blender), with a mortar and pestle, or by cutting the tissue into small pieces. Mechanical disruption is particularly important when using plant cells because they have a tough cell wall. Second, lysis uses detergents and enzymes such as Proteinase K to free the DNA and dissolve cellular proteins.
Step 2: Precipitation
When you complete the lysis step, the DNA has been freed from the nucleus, but it is now mixed with mashed up cell parts. Precipitation separates DNA from this cellular debris. First, Na+ ions (sodium) neutralize the negative charges on the DNA molecules, which makes them more stable and less water soluble. Next, alcohol (such as ethanol or isopropanol) is added and causes the DNA to precipitate out of the aqueous solution because it is not soluble in alcohol.
Step 3: Purification
Now that DNA has been separated from the aqueous phase, it can be rinsed with alcohol to remove any remaining unwanted material and cellular debris. At this point the purified DNA is usually re-dissolved in water for easy handling and storage.
For further lab work, it is important to know the concentration and quality of the DNA. Optical density readings taken by a spectrophotometer can be used to determine the concentration and purity of DNA in a sample. Alternatively, gel electrophoresis can be used to show the presence of DNA in your sample and give an indication of its quality.
As earlier stated, above is the basic/general method of extraction but tends more towards the genomic DNA extraction method. In more details;
Isolating genomic and plasmid DNA for further investigation and downstream application (e.g. PCR, sequencing, etc.) requires totally different protocols. While isolating genomic DNA merely requires you to crack open the cell walls and purify the resulting sample, extracting plasmid DNA may be a bit trickier and more complicated than this. Here’s a rundown on how these techniques differ.
As mentioned earlier, extracting genomic DNA is a simple affair. All one needs to do is disrupt the cell wall using lysozome (an enzyme that effectively hydrolyzes the peptidoglycan component of the cell wall) and proteinase K (a serine protease used in digesting proteins and removing contaminants). The antibacterial enzyme lysostaphin may also be used when working with certain gram-positive species to further enhance enzymatic digestion.
In the same manner, samples may also be subjected to physical and/or mechanical methods to release the genomic DNA from the cell lysate. Most researchers consider bead beating as the lysis method of choice since it is generally faster and more thorough than enzymatic lysis. However, for tough filamentous fungi such as Aspergillus and Fusarium, the cellular materials are best extracted by freezing the sample in liquid nitrogen before using other physical or mechanical methods.
Once the genomic DNA has been successfully extracted, a phenol-chloroform mixture or a suitable protease is used to remove the lipid membranes from the solution. The unwanted contaminants can also be removed by precipitating them with sodium or ammonium acetate.
Plasmid DNA Extraction
While gDNA extraction is pretty straightforward, extracting plasmid DNA can be a little more complicated since you should be able to identify and use the appropriate lysis method to successfully separate the plasmid DNA from the gDNA. Basically, a milder treatment (i.e. alkaline lysis) is required when extracting plasmid DNA. Here’s how you go about extracting them.
Cell Cultivation
The procedure starts with the cultivation of bacterial cells in varying amounts of growth medium. When sufficient growth is achieved, you can remove the cells from the medium through centrifugation.
Resuspension and Cell Lysis
A cell pellet from the saturated culture is resuspended in an isotonic solution containing Tris, EDTA (to disrupt the cell wall and prevent DNases from damaging the plasmid), glucose (to prevent the cells from bursting) and RNase A (to degrade cellular RNA during cell lysis). An alkaline solution containing sodium dodecyl sulfate (SDS) is then added to facilitate cell lysis and the complete denaturation of both genomic and plasmid DNA along with all the proteins in the solution.
Neutralization, Cleaning and Concentration
A potassium acetate solution is then used to neutralize the sample and separate the plasmid DNA from the gDNA. The smaller plasmid DNA tends to renature easily while the larger, more complicated gDNA remains denatured and precipitates out of the solution.
Upon centrifugation, gDNA will form a pellet while plasmid DNA remains soluble. The plasmid DNA remaining in the supernatant can then be precipitated with ethanol or purified using a phenol-chloroform mixture or spin filter technology.